This invention relates to gene therapy methods for treating hemoglobinopathies.
The major form of adult human hemoglobin, HbA, consists of a tetramer of two xcex1-globin chains and two xcex2-globin chains ((xcex12xcex22). Hemoglobinopathies, such as sickle cell anemia and xcex2O-thalassemia, are caused by a failure to produce normal levels of xcex2-globin. Hemoglobin A2 (HbA2), which consists of a tetramer of two xcex1-globin chains and two xcex4-globin chains (xcex12xcex42), is produced in low amounts in most sickle cell patients and in normal adults (2-3% of total hemoglobin) (Steinberg et al., Blood 78:2165, 1991). HbA2 is a potent inhibitor of the sickle hemoglobin (HbS; xcex12xcex2S2) polymerization characteristic of sickle cell anemia (Nagel et al., Proc. Natl. Acad. Sci. USA 76:670, 1979), and shares some functional activity with HbA.
We have shown that modification of the xcex4-globin gene promoter to include a binding site for the erythroid krxc3xcppel-like factor (EKLF) polypeptide that binds to the xcex2-globin gene promoter (hereinafter xe2x80x9cxcex2-EKLFxe2x80x9d) results in increased expression from the xcex4-globin promoter. A modified xcex2-EKLF (hereinafter xe2x80x9cxcex4-EKLFxe2x80x9d) that binds to the wild type xcex4-globin gene promoter can thus be used to induce xcex4-globin expression.
Accordingly, in one aspect, the invention features a method of inducing xcex4-globin gene expression in a cell, such as an erythrocyte precursor cell (e.g., an erythrocyte burst-forming cell (BFC-E) or an erythrocyte colony-forming cell (CFC-E)) or an erythrocyte. In this method, a nucleic acid encoding a xcex4-EKLF polypeptide is introduced into the cell, or a precursor of the cell. Cells into which the nucleic acid can be introduced include erythrocyte precursors, such as BFC-E and CFC-E. Preferably, the nucleic acid is introduced into a pluripotent hematopoietic stem cell, which is capable of self renewal, and thus, in the context of gene therapy methods (see below), minimizes the number of treatments required.
The cell into which the nucleic acid is introduced can be in a mammal or can be a cell that has been removed from a mammal for introduction of the nucleic acid, after which the cell, or progeny thereof, are introduced into a mammal. Typically, this method is carried out for a human patient. In one particular example, the nucleic acid can be introduced into a hematopoietic stem cell that has been obtained from a patient. Induction of xcex4-globin gene expression by this method can be used in gene therapy methods for the treatment of hemoglobinopathies, such as sickle cell anemia and xcex20-thalassemia.
The invention also features xcex4-EKLF polypeptides, which are identical to xcex2-EKLF, except that they contain one or more modifications that permit them to bind to double stranded DNA containing the sequence 5xe2x80x2-TGA AAC CCT-3xe2x80x2 or the sequence 5xe2x80x2-CTA ATG AAA-3xe2x80x2. The modifications that generate xcex4-EKLF polypeptides are generally in a DNA-binding amino acid of a zinc finger of the xcex2-EKLF polypeptide. For example, modifications can be made in any of amino acids -1 and/or 2-6 in any of the three xcex2-EKLF zinc fingers (see below and FIG. 2).
Preferably, the xcex4-EKLF polypeptide of the invention is in a substantially pure preparation. By xe2x80x9csubstantially purexe2x80x9d is meant a preparation that is at least 60% by weight (dry weight) a xcex4-EKLF polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight a xcex4-EKLF polypeptide. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Also featured in the invention is a nucleic acid, such as a nucleic acid containing deoxyribonucleotides (DNA), ribonucleotides (RNA), or combinations or modifications thereof, encoding xcex4-EKLF polypeptides. Preferably, the nucleic acid is in the form of purified DNA. By xe2x80x9cpurified DNAxe2x80x9d is meant DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. The term thus includes, for example, a recombinant DNA molecule that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA molecule that is part of a hybrid gene encoding additional polypeptide sequence.
The invention also includes cells, such as hematopoietic stem cells or erythrocyte precursor cells, e.g., BFC-E or CFC-E, that contain nucleic acids encoding xcex4-EKLF polypeptides.
Vectors containing nucleic acids encoding xcex4-EKLF polypeptides are also included in the invention. The vectors of the invention include those that can be used in gene therapy methods for treating hemoglobinopathies, such as sickle cell anemia and xcex20-thalassemia. For example, adeno-associated viral (AAV) vectors and retroviral vectors (e.g., moloney murine leukemia viral vectors) can be used in the invention. Vectors that can be used for amplifying nucleic acids encoding xcex4-EKLF polypeptides in bacteria are also included in the invention. Preferably, the nucleic acids encoding xcex4-EKLF polypeptides are operably linked to a promoter, for example, the xcex2-globin promoter, or a non-tissue specific promoter, such as the Cytomegalovirus promoter. By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s), such as a promoter, are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins or proteins which include transcriptional activation domains) are bound to the regulatory sequence(s).
The invention also features methods for identifying xcex4-EKLF polypeptides. In these methods, a nucleic acid containing either the sequence 5xe2x80x2-TGA AAC CCT-3xe2x80x2 or the sequence 5xe2x80x2-CTA ATG AAA-3xe2x80x2 is contacted with a candidate polypeptide that has been modified such that it differs from wild type xcex2-EKLF by at least one amino acid, for example, an amino acid in a xcex2-EKLF zinc finger that binds DNA. xcex4-EKLF polypeptides are then identified by their ability to bind to this nucleotide sequence.
Expression of therapeutic levels of gene products in gene therapy methods is often difficult to achieve. Transcription factors are generally required in lower quantities than other types of therapeutic gene products. Thus, an advantage of the present invention is that it provides a gene therapy method involving expression of a transcription factor, xcex4-EKLF.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.